Angiotensin converting enzyme (peptidyldipeptide hydrolase, hereinafter referred to as ACE) occupies a central role in the physiology of hypertension. The enzyme is capable of converting the decapeptide angiotensin I, having the sequence
AspArgValTyrIleHisProPheHisLeu PA1 Arg=arginine PA1 Asp=aspartic acid PA1 Boc=t-butyloxycarbonyl PA1 Cbo=carbobenzyloxy PA1 &lt;Glu=L-pyroglutamic acid PA1 Gly=glycine PA1 Hip=Hippuric acid (Benzoyl-glycine) PA1 His=histidine PA1 Ile=isoleucine PA1 Leu=leucine PA1 Phe=phenylalanine PA1 Pro=proline PA1 .DELTA.Pro=3,4-dehydroproline PA1 Ser=serine PA1 Tos=Tosyl PA1 Trp=tryptophan PA1 Tyr=tyrosine PA1 Val=valine PA1 Pht=phthaloyl PA1 ACE=angiotensin converting enzyme PA1 Hepes=N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid PA1 (ii) aralkylene wherein the alkyl group is 1-6 carbons or alkylaryl; PA1 (iii) phenyl; PA1 (iv) alkylaralkylene wherein the alkyl groups may be the same or different and are 1-6 carbons in length; PA1 (v) substituted alkylene, substituted branched chain alkyl, substituted cycloalkylalkylene, substituted alkyl cycloalkylalkylene, substituted alkylcycloalkylene, substituted alkylaryl, substituted aralkylene, substituted phenyl or substituted alkylaralkylene wherein the substituent or substituents may be the same or different, may be included in an alkylene chain or pendent thereto, and are selected from amino, halo, hydroxy, mercapto, NO.sub.2, carboxy, CONH.sub.2, lower alkyl, halomethyl, hydroxymethyl, aminomethyl, dihalomethyl, trihalomethyl, cyano, mercaptomethyl, methoxymethyl, methylthiomethyl, methoxycarbonylmethyl, cyanomethyl, benzyl, acetoxymethyl, CH.sub.2 .dbd.CH--CH.sub.2 --, isobutyl, mercaptoalkyl of 2-3 carbon atoms, hydroxyalkyl of 2-3 carbon atoms, acetylthioethyl, benzamido, acetamido, phthaloylaminoalkylene wherein the alkylene group has 1-4 carbon atoms, -alkoxycarbonyl isoalkylene wherein the alkyl group contains 1-5 carbons and the isoalkylene group contains 3- 5 carbons, benzoylamino, alkanoylamino of 1-5 carbons, alkylamide of 1-5 carbons, phenylamine, alkylamine of 1-5 carbons, lower alkoxy, aryloxy, lower alkylamino, diloweralkylamino, acylamino, arylamino, guanidino, imidazolyl, indolyl, lower alkylthio, arylthio, carboxy amido and carbolower alkoxy; PA1 (vi) alkylenethio- or alkylenethioalkylene of 1-6 carbons, alkylthioalkylene of 1-6 carbons; PA1 (vii) alkyleneoxy or alkyleneoxyalkylene wherein the alkyl groups may be the same or different and are 1-6 carbons; PA1 (viii) alkoxyphenyl or alkoxybenzyl in which the alkoxy group has 1-3 carbons, phenoxyphenyl, phenoxybenzyl, benzyloxybenzyl or benzyloxyphenyl or a thioether analog of any of them; PA1 (ix) ##STR8## wherein n=0-4, m=0-4, and B=H or a 1-5 carbon alkyl group; or an --SB analog thereof; PA1 (x) ##STR9## wherein n and m have the same significance as above, Y is phenyl, benzyl or a 1-5 carbon alkyl group; PA1 (xi) ##STR10## wherein T and W may be the same or different and are alkylene, aryl, benzyl or cycloalkyl, and P and Q may be the same, or one of them may be H or they may combine to form a ring with the nitrogen to which they are attached. PA1 (ii) hydroxyphenyl or hydroxyphenyl-(1-6C)-alkylene or a thiol analog of either; PA1 (xxviii) alkylene or alkenyl groups of 1-6 carbons substituted with one of the heterocyclic rings from (xxvii) above; PA1 2-amino adipic acid 1-ethyl ester PA1 2-amino pimelic acid 1-ethyl ester PA1 2-amino suberic acid 1-ethyl ester PA1 2-amino azelaic acid 1-ethyl ester PA1 2-amino sebacic acid 1-ethyl ester PA1 oxalacetic acid PA1 ketomalonic acid PA1 para-carboxy phenyl pyruvic acid PA1 indole-1-carboxy-3-pyruvic acid PA1 2-ketoadipic acid
to an octapeptide, angiotensin II, by removal of the carboxy-terminal HisLeu. The symbols for the foregoing chemical moieties and others used throughout this application are explained in the following table:
In each instance the symbol for any amino acid is also used herein at times to refer to a mono-or-di-valent radical of such acid and those of ordinary skill in the art will readily understand the context of each specific use.
Angiotensin I is formed by the action of the enzyme renin, an endopeptidase found in kidney, other tissues, and plasma, on a serum .alpha.-2 globulin.
Blood pressure is affected by certain peptides found in the blood. One of these, angiotensin II, is a powerful pressor (blood pressure elevating) agent. Another, bradykinin, a nonapeptide with the sequence ArgProProGlyPheSerProPheArg is a powerful depressor (blood pressure lowering) agent. In addition to a direct pressor effect, angiotensin II stimulates release of aldosterone which tends to elevate blood pressure by causing retention of extracellular salt and fluids. Angiotensin II is found in measurable amount in the blood of normal humans. However, it is found at elevated concentrations in the blood of patients with renal hypertension.
The level of ACE activity is ordinarily in excess, in both normal and hypertensive humans, of the amount needed to maintain observed levels of angiotensin II. However, it has been found that significant blood pressure lowering is achieved in hypertensive patients by treatment with ACE inhibitors. [Gavras, I. et al., New Engl. J. Med. 291, 817 (1974)].
ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of the penultimate peptide bond at the C-terminal end of a variety of acylated tripeptides and larger polypeptides having an unblocked .alpha.-carboxyl group. The action of ACE results in hydrolytic cleavage of the penultimate peptide bond from the carboxyl-terminal end yielding as reaction products a dipeptide and a remnant.
The reactivity of the enzyme varies markedly depending on the substrate. At least one type of peptide bond, having the nitrogen supplied by proline, is not hydrolyzed at all. The apparent Michaelis constant (Km) varies from substrate to substrate over several orders of magnitude. For general discussion of the kinetic parameters of enzyme catalyzed reactions, see Lehninger, A., Biochemistry, 2nd Ed., Worth Publishers, Inc., New York, 1975, pp. 189-195. Many peptides which are called inhibitors of the enzymatic conversion of angiotensin I to angiotensin II are in fact substrates having a lower Km than angiotensin I. Such peptides are more properly termed competitive substrates. Examples of competitive substrates include bradykinin, and the peptide BPP.sub.5a (also called SQ20475) from snake venom, whose sequence is &lt;GluLysTrpAlaPro.
Numerous synthetic peptide derivatives have been shown to be ACE inhibitors by Ondetti, et al. in U.S. Pat. No. 3,832,337, issued Aug. 27, 1974.
The role of ACE in the pathogenesis of hypertension has prompted a search for inhibitors of the enzyme that could act as antihypertensive drugs. See for example U.S. Pat. Nos. 3,891,616, 3,947,575, 4,052,511 and 4,053,651. A highly effective inhibitor, with high biological activity when orally administered, is D-3-mercapto-2-methylpropanoyl-L-proline, designated SQ14225, or "captopril" disclosed in U.S. Pat. No. 4,046,889 to Ondetti et al., issued Sept. 6, 1977, and in scientific articles by Cushman, D. W. et al., Biochemistry 16, 5484 (1977), and by Ondetti, M. et al., Science 196, 441 (1977). The inhibitor SQ14225 reportedly has an I.sub.50 value of 2.3.times.10.sup.-8 M. The I.sub.50 value reported by Cushman, et al., supra is the concentration of inhibitor required to produce 50% inhibition of the enzyme under a standard assay system containing substrate at a level substantially above K.sub.m. It will be understood that I.sub.50 values are directly comparable when all potential factors affecting the reaction are kept constant. These factors include the source of enzyme, its purity, the substrate used and its concentration, and the composition of the assay buffer. All I.sub.50 data reported herein have been performed with the same assay system and same enzyme (human urinary ACE) and with the same level of substrate and are therefore internally consistent.
The mode of action of SQ14225 has been based upon a model of the active site of ACE developed by analogy with the better known related enzyme, carboxypeptidase A. The active site was postulated to have a cationic site for binding the carboxyl end group of the substrate and a pocket or cleft capable of binding the side chain of the C-terminal amino acid and providing especially tight binding for the heterocyclic ring of a terminal proline residue. A similar pocket for the penultimate amino acid residue was postulated, and the published data suggested a rather stringent steric requirement, since the D-form of the inhibitor was substantially more potent than its stereoisomer or the 3-methyl and unsubstituted analogs. The sulfhydryl group on the inhibitor, postulated to be bound at the active site near the catalytic center, was believed to play a central role in inactivation of the enzyme by combining with the zinc moiety known to be essential for catalytic activity. Substituents on the sulfhydryl, such as a methyl group, and a S-acetyl derivative, substantially reduced potency of the inhibitor. See Cushman, D.W. et al., Biochemistry, supra.
In vitro study of the mechanism by which SQ14225 and its analogs act to inhibit ACE has been somewhat hampered by the instability of these molecules under ambient conditions. For example, it has been observed that a fresh aqueous solution of concentration, e.g., 1 mg per ml of SQ14225 at a pH of about 8 becomes substantially less active upon standing for as little as 30 minutes, and that activity continues to decrease as the solution stands for longer periods. It is believed that this loss in activity is mainly the result of dimerization of SQ14225 occurring at the sulfhydryl end groups, whereby a disulfide is formed which is largely inactive as an inhibitor. Since the free sulfhydryl group is highly reactive and may be readily oxidized to polar acidic moieties such as sulfone and sulfoxide groups, it may also be that the observed in vitro loss of activity of aqueous solutions of SQ14225 on standing is in some part a consequence of one or more such oxidation reactions, with formation of a sulfone or sulfoxide which does not function effectively as an inhibitor for ACE.
Such reports of SQ14225 clinical testing as are currently available, some of which refer to the compound under the name "Captopril" or "Capoten", suggest that the product is sufficiently stable in the normal gastric and intestinal environments of most patients to be an effective inhibitor of ACE when administered orally. It is not yet clear, however, whether there may be a group of patients for which SQ14225 is substantially ineffective. Because of the high reactivity of the free sulfhydryl group, SQ14225 could readily form mixed disulfides with serum, cellular proteins, peptides or other free sulfhydryl group-containing substances in the gastric or intestinal environments, in addition to the possibility for dimer formation or oxidative degradation reactions. A mixed disulfide with protein may be antigenic and, indeed, occasional allergic reactions have been clinically observed. See Gavras, et al., New England J. Med. 298, 991 (1978). Disulfides and oxidative degradation products of SQ14225, if formed, may at best be expected to be largely ineffective as inhibitors. It may be postulated accordingly that dose response to SQ14225 may vary with conditions of administration and among individual patients. Moreover, in at least some patients, unwanted side effects may occur and maintenance of an effective concentration of the inhibitor in the body may be difficult to control.
Adverse effects of SQ14225 in man include fevers and rashes. (Gavras et al., supra). Hoorntje et al., The Lancet i., 1212-1214 (1980) describe the performance of renal biopsies on 13 patients treated with SQ14225. All biopsies showed evidence of immune complex deposition along the glomerular basement membranes, although 9 of 13 patients were asymptomatic at the time of the biopsy. These authors also discussed similarities of their findings with those induced by another drug with a free mercapto group, D-penicillamine.
In an effort to devise better inhibitors of angiotensin converting enzyme that are more stable than captopril and less likely to induce D-pencillamine-like adverse effects, applicants have prepared a series of compounds having side chain structure analogous to an effective substrate for the enzyme, benzoyl-Phe-Ala-Pro, and disclosed them in copending U.S. application Ser. No. 187,992 filed Sept. 17, 1980 (abandoned). Also relevant are the class of carboxylalkyldipeptide derivatives disclosed in European published application of Patchett et al., published on or about June 25, 1980. The present application defines compounds such as N-[L-1-carboxy-3-(carboanilide)propyl]-D,L-Ala-L-Pro, N-[L-1-carboxy-3-(carbo-4-iodoanilide)propyl]-D,L-Ala-L-Pro, and analogs i.e., amides and imides of N-(lower alkylene) Ala-Pro. These two named compounds were found to be unexpectedly effective in inhibiting angiotensin converting enzyme in vitro, that is they have a very low I.sub.50, in the order of 10.sup.-9 M. In contrast, another closely related analog of the two named compounds, i.e., N-[L-1-carboxy-2-(carbopyrrolide)ethyl]-D,L-Ala-Pro, was found to have a much higher I.sub.50, in the order of 10.sup.-7 M, a potency of inhibitor likely to be too low for antihypertensive effectiveness. It is believed, therefore, that amides and imides of N-(lower alkylene)-Ala-Pro and related compounds have unpredictable effects on angiotensin converting enzyme.
In addition, the removal of iodine from N-[L-1-carboxy-3-(carbo-4-iodoanilide)propyl]-D,L-Ala-L-Pro increases intravenous effectiveness three-fold, an unexpectedly large difference in the in vitro effect of the anti-hypertensive compounds of this invention. Hence, amides and imides of N-(lower alkylene)-D,L-Ala-Pro and related compounds are new agents with surprising effectiveness in lowering blood pressure in vivo.
Moreover, since the compounds of this invention do not have the free sulfhydryl group of SQ14225, they are most likely to be stable and have durations of action much longer than that of SQ14225. Thus, inhibitors of this invention may be used for treating hypertension with less frequent dosage schedules than required for SQ14225 and may be capable of administration under less rigorously controlled conditions.